1. Field of the Invention
The present invention is in the field of genetic engineering and molecular biology. It concerns production of proteins, specifically the restriction endonucleases SstI and SacI, in a heterologous organism from a gene carried by a recombinant DNA molecule.
2. Background of the Invention
There has been much effort to clone restriction-modification systems. The first cloning of a DNA endonuclease gene was described by Mann, M. B. et al., Gene 3:97-112 (1978). Since then more than seventy DNA methylase and restriction endonucleases have been cloned, the majority of the restriction endonuclease genes being closely linked to its corresponding methylase gene. Cloning of such genes allows one to produce large quantities of an enzyme.
Several methods by which restriction-modification systems can be cloned have been described. A number of endonuclease and methylase genes have been cloned from endogenous plasmids: EcoRII (Kosykh, V. B. et al., Mol. Gen. Genet. 178:717-718 (1980)), EcoRI (Newman, A. K. et al., J. Biol. Chem. 256:2131-2139 (1981)), Greene, P. J. et al., J. Biol. Chem. 256:2143-2153 (1981)), EcoRV (Bougueleret, L. et al., Nucl. Acids Res. 12:3659-3676 (1984)), PvuII (Blumenthal, R. M. et at., J. Bacteriol.164:501-509 (1985)), and PaeR71 (Gingeras & Brooks, Proc. Natl. Acad. Sci. USA 80:402-406 (1983)). Other methods of cloning include a phage restriction method in which bacterial cells carrying cloned restriction and modification genes will survive phage infection (Mann et al. supra; Walder, R. Y. et al., Proc. Natl. Acad. Sci. USA 78:1503-1507 (1981); Rodicio & Chater, Mol. Gert. Genet. 213:346-353 (1988)), and a procedure based on methylation protection suggested by Mann et at., supra, and Szomolanyi, E. et al., Gene 10:219-225 (1980). This latter scheme involves digestion of a plasmid library with the restriction enzyme to be cloned so that only plasmids whose sequences are modified, because of the presence of the methylase, will produce transformants in a suitable host. This selection has worked well to clone endonuclease and methylase genes together as well as methylase genes alone (Szomolanyi et at., supra; Janulaitis, A. et at., Gene 20:197-204 (1982); Walder, R. Y. et at., J. Biol. Chem. 258:1235-1241 (1983); Kiss & Baldanf, Gene 21:111-119 (1983); Wilson, G. G., Gene 74:281-289 (1988)). However, this technique sometimes yields only the methylase gene, even though the endonuclease and modifying genes are closely linked, as in the case of KpnI (Hammond, A. W. et at., Gene 97:97-102 (1990)).
Cloning of certain restriction-modification systems in E. coli, including DdeI (Howard, K. A. et al., Nucl. Acids Res. 14:7939-7950 (1989)), BamHI (Brooks, J. E. et al., Nucl. Acids Res. 17:979-997 (1989)), KpnI (Hammond, A. W. et at., supra) has required a multi-step approach. In each case, protection of the host with methylase expressed on a plasmid was necessary to stabilize a compatible vector containing the functional endonuclease gene. A head-start model to explain why some restriction-modification systems must be cloned utilizing a protected host was proposed by Wilson, supra. This model states that in order to establish a plasmid carrying a restriction-modification system, methylase protection must be faster than endonuclease digestion. Otherwise, the restriction enzyme would cleave unmethylated plasmid and/or genomic DNA and degrade the plasmid and/or kill the host. Although this model is a plausible explanation of plasmid establishment, it has not been determined previously whether continued independent expression of methylase from a separate plasmid is necessary to maintain the plasmid carrying the restriction-modification system during cell growth and replication.
Restriction endonucleases are named according to the names of the microorganisms that produce them. Microorganisms, in turn, have been named according to a wide variety of criteria, including morphology, biochemical characteristics and 16S ribosomal RNA patterns. Occasionally, two microorganisms are given different names when they are actually very similar if not identical organisms. A Streptomyces stanford strain which makes restriction enzymes SstI and SstII (ATCC29415), and a Streptomyces achromogenes strain (ATCC12767) which makes restriction enzymes SacI and SaclI have identical chromosomal digestion patterns in the area of the SstI locus (see below) and indistinguishable overall digestion patterns when digested with each of several restriction enzymes. It thus appears that they are very similar, if not identical organisms and that the SstI locus and the SacI locus are either very similar or identical.